Protein conformation can be influenced by lipid membranes. This fact seems to be an essential contributor to the function of the HIV accessory protein Nef. Association with the plasma membrane is required for Nef to downregulate CD4 and MHC receptors, thereby enhancing the infectivity of HIV and contributing to AIDS progression. Fully functional Nef appears to require transition from a solution conformation to a membrane-associated conformation. Despite its obvious disease importance, almost nothing is known about the conformation of Nef at the membrane nor about any of Nef's conformational transitions. Although there is information about the solution conformation of Nef deletion variants, functionally important loops and regions were excised to make the protein compatible with NMR and crystallography. If structural details for membrane-associated and full-length Nef could be obtained, the fundamental biochemistry governing Nef interactions and subsequent biological effects would be better understood. Such a knowledge base would ultimately contribute to the rational design of agents that interfere with Nef cellular functions and potentially limit the development of and progression to AIDS. Alternative methods to investigate the membrane-associated conformation of proteins such as Nef include hydrogen exchange mass spectrometry (HXMS) and neutron reflectometry (NR). Because HXMS and NR can be performed with small amounts of dilute protein and in the presence of lipid membranes, conditions not possible with most previous biophysical analyses of full-length Nef, we hypothesize that use of these methods will yield previously unattainable conformational information. Three specific aims will be undertaken: (1). Understand the solution conformation of full-length Nef. HXMS will be performed on Nef from different HIV strains. Missing conformational information about full- length Nef including details of the deleted regions and the effects of sequence variability on Nef conformation will be obtained. (2). Ascertain if and how myristoylated Nef conformation is different in solution. HXMS of myristoylated Nef will be compared to that of non-myristoylated Nef. The regions of conformational alteration upon myristoylation will be determined. (3). Understand the conformation of Nef at the membrane. NR and HXMS will be used to probe the conformation of full-length Nef when associated with membranes and when bound to membrane-anchored target proteins such as tyrosine kinases. The overall shape of Nef at the membrane will be determined and conformational changes upon binding will be ascertained. Taken together, these Aims are expected to provide substantial conformational information about a membrane-associated protein that has been previously very difficult to obtain. As Nef is essential for the infectivity of HIV, this fundamental knowledge is expected to be directly applicable towards the future development of therapeutics. PUBLIC HEALTH RELEVANCE: The focus of this proposal is on a protein made by the human immunodeficiency virus (HIV) called Nef. Nef is essential for viral infectivity. The studies proposed here will determine information about the shape of this protein, especially as it interacts with the plasma membrane of cells. This basic knowledge is essential for the development of therapeutic agents that can interfere with Nef function, thereby preventing HIV infections from causing AIDS.